Modified hirudin proteins and T-cell epitopes in hirudin

ABSTRACT

Modified forms of hirudin having improved properties are disclosed. The modified hirudin compounds are hirudin variants comprising amino acid substitutions in the sequence of hirudin. Peptide molecules consisting of the amino acid residue sequence CILGSDGEKNQCVTGEGTPKPESHNDGDFE (SEQ ID NO: 1) or a sequence consisting of at least 9 consecutive amino acid residues of SEQ ID NO: 1 having a potential MHC class II binding activity are disclosed. The peptide has a stimulation index of &gt;1.8 in a biological assay of cellular proliferation, in which the index is defined as the value of cellular proliferation scored following stimulation by the peptide and divided by the value of cellular proliferation scored in control cells that have not been exposed to the peptide.

This application is the National Stage of International Application No.PCT/EP2004/006943, filed on Jun. 25, 2004. This application claimspriority benefit of European Patent Application No. 03014332.5, filed onJun. 26, 2003.

FIELD

The invention concerns hirudin and in particular modified forms ofhirudin with improved properties. The improved proteins contain aminoacid substitutions at specific positions. The invention providesmodified hirudin with improved biological activity concomitant withreduced immunogenic potential in the protein. The improved proteins areintended for therapeutic use in the treatment of diseases in humans.

BACKGROUND

Native hirudin constitutes a group of nearly identical polypeptides witha molecular weight around 7000 Daltons and consisting of 65 to 66 aminoacids that are produced in the salivary glands of the leech Hirudomedicinalis [Markwardt F (1970) Methods in Enzymology; 19: 924-932].Hirudin is the most potent natural inhibitor of the serine proteasethrombin, with a Ki of 10⁻¹¹ to 10⁻¹⁴ M. It forms a 1:1 complex at theactive site of thrombin resulting in inhibition of its proteolyticactivity [Johnson P H, et al (1989) Sem. Thromb. Hemost.; 15: 320-315;Stone S R, & Hofsteenge J. (1986) Biochemistry; 4622-46242,3]. The X-raycrystal structure of hirudin and the hirudin/thrombin complex have beensolved. The globular N-terminal domain of hirudin binds to theactive-site cleft of thrombin, while the C-terminal tail binds to thefibrinogen binding exosite of thrombin. Site directed mutagenesisstudies have been carried out with both hirudin and the hirudin/thrombincomplex [Grütter M G et al (1990) EMBO Journal; 9: 2361-2365; Rydel T J,et al (1990) Science; 249:277-280; Rydel T J, et al (1991) Journal ofMolecular Biology; 221: 583-601].

Recombinant hirudins (r-hirudin) differ from naturally occurring hirudinby the lack of a sulphate group at tyrosine 63 and the first twoN-terminal amino acids [Märki W E, et al (1997) Semin. Thromb. Haemost;17: 88-93]. Preclinical and clinical studies have shown that r-hirudinis an effective anticoagulant for prevention and treatment of venousthromboembolism in patients undergoing elective hip surgery and forpatients with chronic stable coronary heart disease, acute myocardialinfarction, unstable angina pectoris as an adjunct to coronaryangioplasty, and in combination with intracoronary thrombolysis [LieberV, et al (2002) Seminars in Thrombosis and Hemostasis; 28: 483-489].Recent studies have also suggested that hirudin could be used in stroketherapy [Karabiyikoglu M, et al (2004) Journal of Cerebral Blood Flow &Metabolism. 24: 159-166].

The therapeutic potential of hirudin based thrombin inhibitors has beendemonstrated by the development of Desirudin [Iprivask, Aventis Inc.];[Val¹, Val²]-63-desulphohirudin and Lepirudin [Refludan,Schering/Berlex]; [Leu¹, Thr²]-63-desulphohirudin.

Desirudin was first approved in the EU for thrombosis prophylaxis afterorthopaedic hip and knee surgery. In April 2003 it was approved by theFDA and is now marketed as Iprivask. Lepirudin is approved in both theEuropean Union (1997) and the United States (1998) for the treatment ofpatients suffering from heparin-induced thrombocytopaenia (HIT)[Greinacher A, et al (1999). Circulation; 99: 73-80; Greinacher A,Janssens U, Berg G et al. (1999) Circulation; 99: 587-593; Harenberg J,et al (1997) Seminars in Thrombosis and Hemostasis; 23: 189-196].Heparin is currently used to prevent blood clots in more than 20 millionpatients per year in Europe and the US. In the US and EU alone,approximately 500,000 patients per year are potential candidates forlepirudin therapy.

Despite its evident clinical utility, a significant problem associatedwith the clinical use of hirudin is its immunogenicity. Anti-hirudinantibodies can be detected in patients who have received lepirudin anddesirudin [Song X, et al (1999) Circulation; 100: 1528-1532; Eichler P,et al (2000) Blood; 96: 2373-2378; Greinacher A, et al (2003) Blood;101: 2617-2619; Kischer K-G, et al (2003) Thromb. Haemost; 89: 973-982;Eichler P, et al (2004) Blood; 103: 613-616]. Moreover, lepirudin cancause fatal anaphylaxis, particularly in HIT patients who are treatedwithin 3 months of a previous exposure. Between 1994 and 2002, 9patients have been judged to have had severe anaphylaxis in closetemporal contact with lepirudin. All reactions occurred within minutesof intravenous lepirudin administration, with 4 fatal outcomes. In all 4cases, a previous uneventful treatment course with lepirudin wasidentified (1 to 12 weeks earlier). High-titre IgG anti-lepirudinantibodies were recorded in an additional patient with anaphylaxis[Greinacher A, Lubenow N, Eichler P (2003). Circulation; 108:2062-2065].

Generation of a high affinity long lived antibody response, and inparticular a response that can be seen to have undergone class switchingto high titre IgG antibody, is dependent on CD4+ T-cell help. T cellreceptor (TCR) binding of peptide epitopes presented in the context ofMHC Class II on the surface of antigen presenting cells (APCs), willlead to T-cell proliferation and the production of cytokines that canmodulate immune responses. Patients who develop antibodies to hirudin□have T-cells that are capable of recognising peptide fragments ofhirudin bound to MHC class II molecules.

The present invention is concerned with the identification of T-cellepitopes in hirudin and with hirudin molecules in which amino acidsubstitution and or combinations of substitution have been conducted.The substitutions confer a reduced immunogenic profile on the proteinwhilst retaining functional activity.

SUMMARY OF THE INVENTION

The invention provides hirudin molecules containing amino acidsubstitutions. The amino acid substitutions confer improved propertiesto the protein. The improved properties concern the immunogenicproperties of the protein.

The molecules of the invention have new properties. Such molecules maycause benefit for a patient in need of anticoagulation therapy orprophylaxis.

The molecules of the invention are characterised by the proteinsequences defined herein as M 1-81.

The molecules of the invention are further characterised their relativeactivity in a thrombin inhibition assay of between around 0.2 and around3.3.

The most preferred molecules of the invention is characterised by theprotein sequences M1 and M2 and are further characterised by a relativeactivity of around 1.2-1.4 in a thrombin inhibition assay.

The most preferred molecules of the invention are characterised yetfurther still by comprising sequences demonstrated to show reducedimmunogenicity in human cells. In particular reduced immunogenicity asmeasured using a “T-cell assay” or a “time course assay” as definedherein.

The present invention provides for modified forms of hirudin proteinsthat are expected to display enhanced properties in vivo. The presentinvention discloses the major regions of the hirudin primary sequencethat are immunogenic in man and provides modification to the sequencesto eliminate or reduce the immunogenic effectiveness of these sites.

In one embodiment, synthetic peptides comprising the immunogenic regionscan be provided in pharmaceutical composition for the purpose ofpromoting a tolerogenic response to the whole molecule.

In a further embodiment, the modified hirudin molecules of the presentinvention can be used in pharmaceutical compositions.

In summary the invention is concerned with the following issues:

-   -   A modified hirudin molecule having the biological activity of        hirudin yet containing one or more amino acid substitutions;    -   an accordingly specified molecule, wherein said amino acid        substitutions are conducted within the sequence (A) below        comprising T-cell epitopes wherein;        -   (A)=CILGSDGEKNQCVTGEGTPKPESHNDGDFE (SEQ ID NO: 1);    -   an accordingly specified molecule, wherein said originally        present T-cell epitope sequence (A) comprises MHC class II        ligands or peptide sequences which show the ability to stimulate        or bind T-cells via presentation on class II;    -   a peptide sequence consisting of at least 9 consecutive amino        acid residues as derived from sequence (A) above and its use for        the manufacture of a hirudin like molecule having substantially        no or less immunogenicity than any non-modified molecule and        having the biological activity of hirudin when used in vivo;    -   a pharmaceutical composition comprising 9 or more consecutive        residues from the peptide sequence (A) above;    -   a modified hirudin molecule having the biological activity of        hirudin and comprising one or more amino acid substitutions as        specified above or below;    -   a modified hirudin molecule having the biological activity of        hirudin and comprising at least the amino acid substitutions in        SEQ ID NO: 3: I29A and L30A;    -   a modified hirudin molecule having the biological activity of        hirudin and comprising at least the amino acid substitutions in        SEQ ID NO: 3: I29R and L30H;    -   a modified hirudin molecule of structure (M; SEQ ID NO: 2):

V V Y T D C T E S G Q N X¹ C X² C E G S V X³ C G Q G N K C X⁴ X⁵ G S D GE K N Q C X⁶ T G E G T P X⁷ X⁸ E S H N X⁹ G D X¹⁰ E E I P E E Y L Q

-   -   -   wherein;        -   X¹=T or L        -   X²=T or A or H or Q or T or L;        -   X³=A or G or H or K or N or P or Q or R or V;        -   X⁴=A or D or E or G or H or K or N or Q or R or S or T or I;        -   X⁵=A or D or E or G or H or K or N or P or Q or R or S or T            or L;        -   X⁶=A or T or V;        -   X⁷=T or K;        -   X⁸=A or T or P;        -   X⁹=E or N or R or D;        -   X¹⁰=H or F            and whereby X¹=L, X²=L, X³=V, X⁴=I, X⁵=L, X⁶=V, X⁷=K, X⁸=P,            X⁹=D and X¹⁰=F are simultaneously excluded.

The mutant proteins of the present invention are readily made usingrecombinant DNA techniques well known in the art and the inventionprovides methods for the recombinant production of such molecules.

In as far as this invention relates to modified hirudin, compositionscontaining such modified hiridin proteins or fragments of modifiedhirudin proteins and related compositions should be considered withinthe scope of the invention. In another aspect, the present inventionrelates to nucleic acids encoding modified hirudin entities. In afurther aspect the present invention relates to methods for therapeutictreatment of humans using the modified hirudin.

DETAILED DESCRIPTION OF THE INVENTION

In nature, the mature hirudin protein is polypeptide of 65 to 66 aminoacids. The amino acid sequence of a wild type (WT) hirudin (depicted assingle-letter code) is as follows (M82):

V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C I L G S D G E KN Q C V T G E G T P K P E S H N D G D F E E I P E E Y L Q (SEQ ID NO: 3)

The term “hirudin” is used herein to denote recombinant or syntheticequivalents of the above sequence which are in turn analogues of thenative hirudin proteins constituting a group of nearly identicalpolypeptides with a molecular weight around 7000 Daltons and which areproduced in the salivary glands of the leech Hirudo medicinalis. In someinstances the term is also used more broadly herein to include modifiedhirudin proteins or more especially a hirudin mutein.

The term “mutein” is used herein to denote a hirudin protein engineeredto contain one or more amino acid substitutions differing from the abovenative sequence.

The term “peptide” as used herein, is a compound that includes two ormore amino acids. The amino acids are linked together by a peptide bond.

A peptide bond is the sole covalent linkage between amino acids in thelinear backbone structure of all peptides, polypeptides or proteins. Thepeptide bond is a covalent bond, planar in structure and chemicallyconstitutes a substituted amide. An “amide” is any of a group of organiccompounds containing the grouping —CONH—.

There are 20 different naturally occurring amino acids involved in thebiological production of peptides, and any number of them may be linkedin any order to form a peptide chain or ring. The naturally occurringamino acids employed in the biological production of peptides all havethe L-configuration. Synthetic peptides can be prepared employingconventional synthetic methods, utilizing L-amino acids, D-amino acids,or various combinations of amino acids of the two differentconfigurations. Some peptides contain only a few amino acid units. Shortpeptides, e.g., having less than ten amino acid units, are sometimesreferred to as “oligopeptides”. Other peptides contain a large number ofamino acid residues, e.g. up to 100 or more, and are referred to as“polypeptides”. By convention, a “polypeptide” may be considered as anypeptide chain containing three or more amino acids, whereas a“oligopeptide” is usually considered as a particular type of “short”polypeptide. Thus, as used herein, it is understood that any referenceto a “polypeptide” also includes an oligopeptide. Further, any referenceto a “peptide” includes polypeptides, oligopeptides, and proteins. Eachdifferent arrangement of amino acids forms different polypeptides orproteins. The number of polypeptides—and hence the number of differentproteins—that can be formed is practically unlimited.

Since the peptide bond is the sole linkage between amino acids, allpeptides, polypeptides or proteins have defined termini conventionallyreferred to as the “N-terminus” or “N-terminal” residue and the“C-terminus” or “C-terminal residue”. The N-terminal residue bears afree amino group, whereas the C-terminal residue bears a free carboxylgroup.

The term “T-cell epitope” means according to the understanding of thisinvention an amino acid sequence which is able to bind MHC class II,able to stimulate T-cells and/or also to bind (without necessarilymeasurably activating) T-cells in complex with MHC class II. Inprinciple such an amino acid sequence can be not less than 9 residues inlength but typically will be 10 or 11 or 12 or 13 or 14 or 15 or moreresidues in length.

Reference to “substantially non-immunogenic” or “reduced immunogenicpotential” includes reduced immunogenicity compared to a parent proteinor to a fusion protein containing the wild-type (WT) or native aminoacid sequences of the test moiety.

The term “immunogenicity” includes an ability to provoke, induce orotherwise facilitate a humoral and or T-cell mediated response in a hostanimal and in particular where the “host animal” is a human.

The terms “T-cell assay” and “immunogenicity assay” concern ex vivomeasures of immune reactivity. As such these involve a test immunogene.g. a protein or peptide being brought into contact with live humanimmune cells and their reactivity measured. A typical parameter ofinduced reactivity is proliferation. The presence of suitable controldeterminations are critical and implicit in the assay.

“Time course assay” refers to a biological assay such as a proliferationassay in which determinations of activity are made sequentially over aperiod of time. In the present context, a “time course T-cell assay”,refers to the determination of T-cell proliferation in response to atest immunogen (peptide) at multiple times following exposure to thetest immunogen. The terms “time course T-cell assay” and “time courseimmunogenicity assay” may be used interchangeably herein.

One conventional way in which T-cell assays are expressed is by use of a“stimulation index” or “SI”. The stimulation index (SI) isconventionally derived by division of the proliferation score (e.g.counts per minute of radioactivity if using for example ³H-thymidineincorporation) measured to a test immunogen such as a peptide by thescore measured in cells not contacted with a test immunogen. Testimmunogens (peptides) which evoke no response give SI=1.0 although inpractice SI values in the range 0.8-1.2 are unremarkable. The inventorshave established that in the operation of such immunogenicity assays, astimulation index equal to or greater than 2.0 is a useful measure ofsignificant induced proliferation.

PBMC means peripheral blood mononuclear cells in particular as obtainedfrom a sample of blood from a donor. PBMC are readily isolated fromwhole blood samples using a density gradient centrifugation techniquewell understood in the art and comprise predominantly lymphocytes (B andT cells) and monocytes. Other cell types are also represented.

“Relative activity” means according the present context activitymeasured for a test protein in any single assay expressed relative tothe activity measured for a positive control protein in an identicalassay and usually conducted in parallel. Thus if the test protein andthe control protein have the same measured activity the relativeactivity is said to be 1.

A “thrombin inhibition assay” according to the present context means anin vitro assay able to provide a reading of the functional capability ofthe test protein. In the present instance this means the ability of agiven hirudin mutein to evoke a specific measurable chromogenicresponse. Particularly suitable assays are exemplified herein usingthrombin and the chromogenic thrombin substrate H-D-Phe-Pep-Arg-pNA.Other assay formats can be contemplated to also provide quantitativeestimations of specific activity of the test molecules and permit ED₅₀determinations. Examples include the Activated Partial ThromboplastinTime coagulation (aPTT) assay and the Ecarin clotting time (ECT) assay,each well known in the art.

In another aspect, the present invention relates to nucleic acidsencoding modified hirudin entities. Such nucleic acids are preferablycomprised within an expression vector. The control sequence that aresuitable for prokaryotes, for example, include a promoter, optionally anoperator sequence, and a ribosome binding site. Eukaryotic cells areknown to utilise promoters, enhancers and polyadenylation signals. Suchnucleic acids in general comprise a selection means typically anadditional gene encoding a protein able to provide for the survival ofthe host cell. An example of such a selection gene is the beta-lactamasegene suitable for some E. coli host cells and this and others are wellknown in the art [“Molecular Cloning: A Laboratory Manual”, secondedition (Sambrook et al., 1989); “Gene Transfer Vectors for MammalianCells” (J. M. Miller & M. P. Calos, eds., 1987); “Current Protocols inMolecular Biology” (F. M. Ausubel et al., eds., 1987)].

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apre-sequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in the same reading frame.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

In some embodiments the expression vector comprises a nucleic acidsequence encoding a hirudin variant operably linked to an expressioncontrol sequence. In various embodiments the expression vector comprisesa nucleic acid sequence encoding a protein selected from the groupcomprising inclusively M1 to M81. Such an expression vector willcomprise at least the hirudin encoding domain of one of the saidproteins operably linked with suitable expression control and selectionsequences. Such an expression vector would include degenerate versionsof the nucleic acid wherein degeneracy in relation to polynucleotidesrefers to the fact well recognised that in the genetic code many aminoacids are specified by more than one codon. The degeneracy of the codeaccounts for 20 different amino acids encoded by 64 possible tripletsequences of the four different bases comprising DNA.

Another aspect of the present invention is a cultured cell comprising atleast one of the above-mentioned vectors.

A further aspect of the present invention is a method for preparing themodified hirudin comprising culturing the above mentioned cell underconditions permitting expression of the hirudin from the expressionvector and purifying the hirudin from the cell.

Whilst the hirudin molecules of the invention and are readily made usingthe above described recombinant DNA based methods, it is recognised thatas short polypeptides, the preferred molecules of the invention can bemade by wholly synthetic means using solid phase chemical synthesismethods or equivalent techniques well known in the art [for review seeBruckdorfer, T. et al (2004) Curr. Pharm. Biotechnol. 5:29-43 andreferences therein].

In a yet further aspect, the present invention relates to methods fortherapeutic treatment of humans using the hirudin compositions. Foradministration to an individual, any of the modified compositions wouldbe produced to be preferably at least 80% pure and free of pyrogens andother contaminants. It is further understood that the therapeuticcompositions of the hirudin proteins may be used in conjunction with apharmaceutically acceptable excipient. The pharmaceutical compositionsaccording to the present invention are prepared conventionally,comprising substances that are customarily used in pharmaceuticals, e.g.Remington's Pharmaceutical Sciences, (Alfonso R. Gennaro ed. 18^(th)edition 1990), including excipients, carriers adjuvants and buffers. Thecompositions can be administered, e.g. parenterally, enterally,intramuscularly, subcutaneously, intravenously or other routes useful toachieve an effect. Conventional excipients include pharmaceuticallyacceptable organic or inorganic carrier substances suitable forparenteral, enteral and other routes of administration that do notdeleteriously react with the agents. For parenteral application,particularly suitable are injectable sterile solutions, preferably oilor aqueous solutions, as well as suspensions, emulsions or implants,including suppositories. Ampules are convenient unit dosages. Thepharmaceutical preparations can be sterilised and, if desired, mixedwith stabilisers, wetting agents, emulsifiers, salts for influencingosmotic pressure, buffers or other substances that do not reactdeleteriously with the active compounds.

The major embodiments of the present invention are encompassed by theprotein sequences M 1-81. A number of muteins show improved activityrelative to the WT molecule (M82). All active muteins are embodiments ofthe invention.

The hirudin muteins of the present were constructed to be lessimmunogenic than the parental molecule. The design of individual muteinswas directed from immunological considerations as well as functionalactivity data. A single major region of immunological importance withinthe molecule was defined using screening assays involving use of PBMCpreparations from healthy donor subjects. This approach has proven to bea particularly effective method for the identification such biologicallyrelevant immunogenic peptides and is disclosed herein as an embodimentof the invention. In the present study, the method has involved thetesting of overlapping hirudin-derived peptide sequences in a scheme soas to scan and test the entire hirudin sequence. Such a scan requiredsynthesis and use of 19 peptides each of 15 residues in length. Thesynthetic peptides were tested for ability to evoke a proliferativeresponse in human T-cells cultured in vitro. Where this type of approachis conducted using naïve human T-cells taken from healthy donors, theinventors have established that a stimulation index equal to or greaterthan 2.0 is a useful measure of induced proliferation.

One significant epitope region (termed R1) was identified in thesestudies. R1 encompasses residues 28-57 of SEQ ID NO: 3 and comprises thesequence CILGSDGEKNQCVTGEGTPKPESHNDGDFE (SEQ ID NO: 1).

The R1 peptide sequence represents the critical information required forthe construction of modified hirudin molecules in which the epitope iscompromised. Equally, the R1 sequence represent the critical informationrequired for the production of tolerogenic peptides. Epitope region R1is an embodiment of the invention.

Under the scheme of the present, the epitopes are compromised bymutation to result in sequences no longer able to function as T-cellepitopes. It is possible to use recombinant DNA methods to achievedirected mutagenesis of the target sequences and many such techniquesare available and well known in the art. Broadly, the hirudin muteinsherein were constructed containing mutations within the immunogenicregion R1. Individual residues were targeted based upon the knownbinding properties of HLA-DR molecules in that they have an almostexclusive preference for a hydrophobic amino acid in pocket 1 and thatthis is the most important determinant of peptide binding [Jardetzky, T.S. et al (1990), EMBO J. 9: 1797-1803; Hill, C. M. et al (1994) J.Immunol. 152: 2890-2898]. Exhaustive mutational analysis identifiedthose residues both inside and outside R1 that could be altered withoutadversely affecting the activity of the fusion protein. A number ofmuteins show improved activity relative to the WT molecule. All activemuteins are embodiments of the invention.

The general method of the present invention leading to the modified TPOcomprises the following steps:

(a) determining the amino acid sequence of the polypeptide or partthereof;

(b) identifying one or more potential T-cell epitopes within the aminoacid sequence of the protein by any method including determination ofthe binding of the peptides to MHC molecules using in vitro or in silicotechniques or biological assays;

(c) designing new sequence variants with one or more amino acids withinthe identified potential T-cell epitopes modified in such a way tosubstantially reduce or eliminate the activity of the T-cell epitope asdetermined by the binding of the peptides to MHC molecules using invitro or in silico techniques or biological assays. Such sequencevariants are created in such a way to avoid creation of new potentialT-cell epitopes by the sequence variations unless such new potentialT-cell epitopes are, in turn, modified in such a way to substantiallyreduce or eliminate the activity of the T-cell epitope; and

(d) constructing such sequence variants by recombinant DNA techniquesand testing said variants in order to identify one or more variants withdesirable properties according to well known recombinant techniques.

Taken together, the inventors have been able to define modified hirudinproteins which can be depicted by the following structure (M; SEQ ID NO:2):

V V Y T D C T E S G Q N X¹ C X² C E G S V X³ C G Q G N K C X⁴ X⁵ G S D GE K N Q C X⁶ T G E G T P X⁷ X⁸ E S H N X⁹ G D X¹⁰ E E I P E E Y L Qwherein;

-   -   X¹=T or L    -   X²=T or A or H or Q or T or L;    -   X³=A or G or H or K or N or P or Q or R or V;    -   X⁴=A or D or E or G or H or K or N or Q or R or S or T or I;    -   X⁵=A or D or E or G or H or K or N or P or Q or R or S or T or        L;    -   X⁶=A or T or V;    -   X⁷=T or K;    -   X⁸=A or T or P;    -   X⁹=E or N or R or D;    -   X¹⁰=H or F        and whereby X¹=L, X²=L, X³=V, X⁴=I, X⁵=L, X⁶=V, X⁷=K, X⁸=P, X⁹=D        and X¹⁰=F are simultaneously excluded.

The following, figures, sequence listing and examples are provided toaid the understanding of the present invention. It is understood thatmodifications can be made in the procedures set fourth without departingfrom the spirit of the invention.

DESCRIPTION OF THE SEQUENCES

To aid the understanding of the invention, Table 1 below sets out adescription of the hirudin muteins. The derivation and properties ofthese proteins are also more fully disclosed in the examples. In Table1, the heading labeled “Substitution(s)” refers to substitutions in SEQID NO: 3.

TABLE 1 Description of the sequences M No Substitution(s) M NoSubstitution(s) M 1  I29A L30A M 42 V21A M 2  I29R L30H M 43 V21G M 3 I29E L30K V40A M 44 V21H M 4  I29E L30K V40T M 45 V21K M 5  I29E L30KD53E M 46 V21N M 6  I29E L30K D53N M 47 V21P M 7  I29R L30K V40A M 48V21Q M 8  I29R L30K V40T M 49 V21R M 9  I29R L30K D53E M 50 I29A M 10I29R L30K D53N M 51 I29D M 11 I29R L30R V40T M 52 I29E M 12 I29A L30K M53 I29G M 13 I29A L30Q M 54 I29H M 14 I29A L30R M 55 I29K M 15 I29A L30TM 56 I29N M 16 I29D L30A M 57 I29Q M 17 I29D L30Q M 58 I29R M 18 I29DL30R M 59 I29S M 19 I29E L30K M 60 I29T M 20 I29E L30Q M 61 L30A M 21I29E L30R M 62 L30D M 22 I29E L30T M 63 L30E M 23 I29R L30K M 64 L30G M24 I29R L30Q M 65 L30H M 25 I29R L30R M 66 L30K M 26 I29R L30T M 67 L30NM 27 I29S L30A M 68 L30P M 28 I29S L30K M 69 L30Q M 29 I29S L30Q M 70L30R M 30 I29S L30R M 71 L30S M 31 I29S L30T M 72 L30T M 32 I29T L30A M73 V40A M 33 I29T L30K M 74 V40T M 34 I29T L30Q M 75 K47T M 35 I29T L30RM 76 P48A M 36 I29T L30T M 77 P48T M 37 L13T M 78 D53E M 38 L15A M 79D53N M 39 L15H M 80 D53R M 40 L15Q M 81 F56H M 41 L15T M 82 WT

TABLE A Sequences of modified (M1–M81) and wild-type hirudin (M82), andT-cell epitope containing region A within wild-type hirudin: M 1 (SEQ IDNO: 4) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C A A G S DG E K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 2 (SEQID NO: 5) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C R H GS D G E K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 3(SEQ ID NO: 6) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C EK G S D G E K N Q C A T G E G T P K P E S H N D G D F E E I P E E Y L QM 4 (SEQ ID NO: 7) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N KC E K G S D G E K N Q C T T G E G T P K P E S H N D G D F E E I P E E YL Q M 5 (SEQ ID NO: 8) V V Y T D C T E S G Q N L C L C E G S V V C G Q GN K C E K G S D G E K N Q C V T G E G T P K P E S H N E G D F E E I P EE Y L Q M 6 (SEQ ID NO: 9) V V Y T D C T E S G Q N L C L C E G S V V C GQ G N K C E K G S D G E K N Q C V T G E G T P K P E S H N N G D F E E IP E E Y L Q M 7 (SEQ ID NO: 10) V V Y T D C T E S G Q N L C L C E G S VV C G Q G N K C R K G S D G E K N Q C A T G E G T P K P E S H N D G D FE E I P E E Y L Q M 8 (SEQ ID NO: 11) V V Y T D C T E S G Q N L C L C EG S V V C G Q G N K C R K G S D G E K N Q C T T G E G T P K P E S H N DG D F E E I P E E Y L Q M 9 (SEQ ID NO: 12) V V Y T D C T E S G Q N L CL C E G S V V C G Q G N K C R K G S D G E K N Q C V T G E G T P K P E SH N E G D F E E I P E E Y L Q M 10 (SEQ ID NO: 13) V V Y T D C T E S G QN L C L C E G S V V C G Q G N K C R K G S D G E K N Q C V T G E G T P KP E S H N N G D F E E I P E E Y L Q M 11 (SEQ ID NO: 14) V V Y T D C T ES G Q N L C L C E G S V V C G Q G N K C R R G S D G E K N Q C T T G E GT P K P E S H N D G D F E E I P E E Y L Q M 12 (SEQ ID NO: 15) V V Y T DC T E S G Q N L C L C E G S V V C G Q G N K C A K G S D G E K N Q C V TG E G T P K P E S H N D G D F E E I P E E Y L Q M 13 (SEQ ID NO: 16) V VY T D C T E S G Q N L C L C E G S V V C G Q G N K C A Q G S D G E K N QC V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 14 (SEQ ID NO:17) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C A R G S D GE K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 15 (SEQID NO: 18) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C A T GS D G E K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 16(SEQ ID NO: 19) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K CD A G S D G E K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y LQ M 17 (SEQ ID NO: 20) V V Y T D C T E S G Q N L C L C E G S V V C G Q GN K C D Q G S D G E K N Q C V T G E G T P K P E S H N D G D F E E I P EE Y L Q M 18 (SEQ ID NO: 21) V V Y T D C T E S G Q N L C L C E G S V V CG Q G N K C D E G S D G E K N Q C V T G E G T P K P E S H N D G D F E EI P E E Y Q M 19 (SEQ ID NO: 22) V V Y T D C T E S G Q N L C L C E G S VV C G Q G N K C E K G S D G E K N Q C V T G E G T P K P E S H N D G D FE E I P E E Y L Q M 20 (SEQ ID NO: 23) V V Y T D C T E S G Q N L C L C EG S V V C G Q G N K C E Q G S D G E K N Q C V T G E G T P K P E S H N DG D F E E I P E E Y L Q M 21 (SEQ ID NO: 24) V V Y T D C T E S G Q N L CL C E G S V V C G Q G N K C E R G S D G E K N Q C V T G E G T P K P E SH N D G D F E E I P E E Y L Q M 22 (SEQ ID NO: 25) V V Y T D C T E S G QN L C L C E G S V V C G Q G N K C E T G S D G E K N Q C V T G E G T P KP E S H N D G D F E E I P E E Y L Q M 23 (SEQ ID NO: 26) V V Y T D C T ES G Q N L C L C E G S V V C G Q G N K C R K G S D G E K N Q C V T G E GT P K P E S H N D G D F E E I P E E Y L Q M 24 (SEQ ID NO: 27) V V Y T DC T E S G Q N L C L C E G S V V C G Q G N K C R Q G S D G E K N Q C V TG E G T P K P E S H N D G D F E E I P E E Y L Q M 25 (SEQ ID NO: 28) V VY T D C T E S G Q N L C L C E G S V V C G Q G N K C R R G S D G E K N QC V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 26 (SEQ ID NO:29) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C R T G S D GE K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 27 (SEQID NO: 30) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C S A GS D G E K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 28(SEQ ID NO: 31) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K CS K G S D G E K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y LQ M 29 (SEQ ID NO: 32) V V Y T D C T E S G Q N L C L C E G S V V C G Q GN K C S Q G S D G E K N Q C V T G E G T P K P E S H N D G D F E E I P EE Y L Q M 30 (SEQ ID NO: 33) V V Y T D C T E S G Q N L C L C E G S V V CG Q G N K C S R G S D G E K N Q C V T G E G T P K P E S H N D G D F E EI P E E Y L Q M 31 (SEQ ID NO: 34) V V Y T D C T E S G Q N L C L C E G SV V C G Q G N K C S T G S D G E K N Q C V T G E G T P K P E S H N D G DF E E I P E E Y L Q M 32 (SEQ ID NO: 35) V V Y T D C T E S G Q N L C L CE G S V V C G Q G N K C T A G S D G E K N Q C V T G E G T P K P E S H ND G D F E E I P E E Y L Q M 33 (SEQ ID NO: 36) V V Y T D C T E S G Q N LC L C E G S V V C G Q G N K C T K G S D G E K N Q C V T G E G T P K P ES H N D G D F E E I P E E Y L Q M 34 (SEQ ID NO: 37) V V Y T D C T E S GQ N L C L C E G S V V C G Q G N K C T Q G S D G E K N Q C V T G E G T PK P E S H N D G D F E E I P E E Y L Q M 35 (SEQ ID NO: 38) V V Y T D C TE S G Q N L C L C E G S V V C G Q G N K C T R G S D G E K N Q C V T G EG T P K P E S H N D G D F E E I P E E Y L Q M 36 (SEQ ID NO: 39) V V Y TD C T E S G Q N L C L C E G S V V C G Q G N K C T T G S D G E K N Q C VT G E G T P K P E S H N D G D F E E I P E E Y L Q M 37 (SEQ ID NO: 40) VV Y T D C T E S G Q N T C L C E G S V V C G Q G N K C I L G S D G E K NQ C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 38 (SEQ ID NO:41) V V Y T D C T E S G Q N L C A C E G S V V C G Q G N K C I L G S D GE K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 39 (SEQID NO: 42) V V Y T D C T E S G Q N L C H C E G S V V C G Q G N K C I L GS D G E K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 40(SEQ ID NO: 43) V V Y T D C T E S G Q N L C Q C E G S V V C G Q G N K CI L G S D G E K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y LQ M 41 (SEQ ID NO: 44) V V Y T D C T E S G Q N L C T C E G S V V C G Q GN K C I L G S D G E K N Q C V T G E G T P K P E S H N D G D F E E I P EE Y L Q M 42 (SEQ ID NO: 45) V V Y T D C T E S G Q N L C L C E G S V A CG Q G N K C I L G S D G E K N Q C V T G E G T P K P E S H N D G D F E EI P E E Y L Q M 43 (SEQ ID NO: 46) V V Y T D C T E S G Q N L C L C E G SV G C G Q G N K C I L G S D G E K N Q C V T G E G T P K P E S H N D G DF E E I P E E Y L Q M 44 (SEQ ID NO: 47) V V Y T D C T E S G Q N L C L CE G S V H C G Q G N K C I L G S D G E K N Q C V T G E G T P K P E S H ND G D F E E I P E E Y L Q M 45 (SEQ ID NO: 48) V V Y T D C T E S G Q N LC L C E G S V K C G Q G N K C I L G S D G E K N Q C V T G E G T P K P ES H N D G D F E E I P E E Y L Q M 46 (SEQ ID NO: 49) V V Y T D C T E S GQ N L C L C E G S V N C G Q G N K C I L G S D G E K N Q C V T G E G T PK P E S H N D G D F E E I P E E Y L Q M 47 (SEQ ID NO: 50) V V Y T D C TE S G Q N L C L C E G S V P C G Q G N K C I L G S D G E K N Q C V T G EG T P K P E S H N D G D F E E I P E E Y L Q M 48 (SEQ ID NO: 51) V V Y TD C T E S G Q N L C L C E G S V Q C G Q G N K C I L G S D G E K N Q C VT G E G T P K P E S H N D G D F E E I P E E Y L Q M 49 (SEQ ID NO: 52) VV Y T D C T E S G Q N L C L C E G S V R C G Q G N K C I L G S D G E K NQ C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 50 (SEQ ID NO:53) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C A L G S D GE K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 51 (SEQID NO: 54) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C D L GS D G E K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 52(SEQ ID NO: 55) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K CE L G S D G E K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y LQ M 53 (SEQ ID NO: 56) V V Y T D C T E S G Q N L C L C E G S V V C G Q GN K C G L G S D G E K N Q C V T G E G T P K P E S H N D G D F E E I P EE Y L Q M 54 (SEQ ID NO: 57) V V Y T D C T E S G Q N L C L C E G S V V CG Q G N K C H L G S D G E K N Q C V T G E G T P K P E S H N D G D F E EI P E E Y L Q M 55 (SEQ ID NO: 58) V V Y T D C T E S G Q N L C L C E G SV V C G Q G N K C K L G S D G E K N Q C V T G E G T P K P E S H N D G DF E E I P E E Y L Q M 56 (SEQ ID NO: 59) V V Y T D C T E S G Q N L C L CE G S V V C G Q G N K C N L G S D G E K N Q C V T G E G T P K P E S H ND G D F E E I P E E Y L Q M 57 (SEQ ID NO: 60) V V Y T D C T E S G Q N LC L C E G S V V C G Q G N K C Q L G S D G E K N Q C V T G E G T P K P ES H N D G D F E E I P E E Y L Q M 58 (SEQ ID NO: 61) V V Y T D C T E S GQ N L C L C E G S V V C G Q G N K C R L G S D G E K N Q C V T G E G T PK P E S H N D G D F E E I P E E Y L Q M 59 (SEQ ID NO: 62) V V Y T D C TE S G Q N L C L C E G S V V C G Q G N K C S L G S D G E K N Q C V T G EG T P K P E S H N D G D F E E I P E E Y L Q M 60 (SEQ ID NO: 63) V V Y TD C T E S G Q N L C L C E G S V V C G Q G N K C T L G S D G E K N Q C VT G E G T P K P E S H N D G D F E E I P E E Y L Q M 61 (SEQ ID NO: 64) VV Y T D C T E S G Q N L C L C E G S V V C G Q G N K C I A G S D G E K NQ C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 62 (SEQ ID NO:65) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C I D G S D GE K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M63 (SEQID NO: 66) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C I E GS D G E K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y L Q M 64(SEQ ID NO: 67) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K CI G G S D G E K N Q C V T G E G T P K P E S H N D G D F E E I P E E Y LQ M 65 (SEQ ID NO: 68) V V Y T D C T E S G Q N L C L C E G S V V C G Q GN K C I H G S D G E K N Q C V T G E G T P K P E S H N D G D F E E I P EE Y L Q M 66 (SEQ ID NO: 69) V V Y T D C T E S G Q N L C L C E G S V V CG Q G N K C I K G S D G E K N Q C V T G E G T P K P E S H N D G D F E EI P E E Y L Q M 67 (SEQ ID NO: 70) V V Y T D C T E S G Q N L C L C E G SV V C G Q G N K C I N G S D G E K N Q C V T G E G T P K P E S H N D G DF E E I P E E Y L Q M 68 (SEQ ID NO: 71) V V Y T D C T E S G Q N L C L CE G S V V C G Q G N K C I P G S D G E K N Q C V T G E G T P K P E S H ND G D F E E I P E E Y L Q M 69 (SEQ ID NO: 72) V V Y T D C T E S G Q N LC L C E G S V V C G Q G N K C I Q G S D G E K N Q C V T G E G T P K P ES H N D G D F E E I P E E Y L Q M 70 (SEQ ID NO: 73) V V Y T D C T E S GQ N L C L C E G S V V C G Q G N K C I R G S D G E K N Q C V T G E G T PK P E S H N D G D F E E I P E E Y L Q M 71 (SEQ ID NO: 74) V V Y T D C TE S G Q N L C L C E G S V V C G Q G N K C I S G S D G E K N Q C V T G EG T P K P E S H N D G D F E E I P E E Y L Q M 72 (SEQ ID NO: 75) V V Y TD C T E S G Q N L C L C E G S V V C G Q G N K C I T G S D G E K N Q C VT G E G T P K P E S H N D G D F E E I P E E Y L Q M 73 (SEQ ID NO: 76) VV Y T D C T E S G Q N L C L C E G S V V C G Q G N K C I L G S D G E K NQ C A T G E G T P K P E S H N D G D F E E I P E E Y L Q M 74 (SEQ ID NO:77) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C I L G S D GE K N Q C T T G E G T P K P E S H N D G D F E E I P E E Y L Q M 75 (SEQID NO: 78) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K C I L GS D G E K N Q C V T G E G T P T P E S H N D G D F E E I P E E Y L Q M 76(SEQ ID NO: 79) V V Y T D C T E S G Q N L C L C E G S V V C G Q G N K CI L G S D G E K N Q C V T G E G T P K A E S H N D G D F E E I P E E Y LQ M 77 (SEQ ID NO: 80) V V Y T D C T E S G Q N L C L C E G S V V C G Q GN K C I L G S D G E K N Q C V T G E G T P K T E S H N D G D F E E I P EE Y L Q M 78 (SEQ ID NO: 81) V V Y T D C T E S G Q N L C L C E G S V V CG Q G N K C I L G S D G E K N Q C V T G E G T P K P E S H N E G D F E EI P E E Y L Q M 79 (SEQ ID NO: 82) V V Y T D C T E S G Q N L C L C E G SV V C G Q G N K C I L G S D G E K N Q C V T G E G T P K P E S H N N G DF E E I P E E Y L Q M 80 (SEQ ID NO: 83) V V Y T D C T E S G Q N L C L CE G S V V C G Q G N K C I L G S D G E K N Q C V T G E G T P K P E S H NR G D F E E I P E E Y L Q M 81 (SEQ ID NO: 84) V V Y T D C T E S G Q N LC L C E G S V V C G Q G N K C I L G S D G E K N Q C V T G E G T P K P ES H N D G D H E E I P E E Y L Q M 82 (SEQ ID NO: 3) V V Y T D C T E S GQ N L C L C E G S V V C G Q G N K C I L G S D G E K N Q C V T G E G T PK P E S H N D G D F E E I P E E Y L Q (A) (SEQ ID NO: 1): C I L G S D GE K N Q C V T G E G T P K P E S H N D G D F E

DESCRIPTION OF THE FIGURES

FIG. 1:

The mature sequence of [Val¹ Val²]-hirudin (SEQ ID NO: 3) with regionsof immunogenicity highlighted in bold. The most preferred modificationsare to residues 29 and 30 (underlined). The preferred alternativesubstitutions are indicated above the sequence.

FIG. 2:

Immunogenicity of [Val¹ Val²]-hirudin variant peptides. Two cohorts of20 healthy donors were used to test the immunogenicity of wild typeregion 1 and modified region 1 peptides. Proliferation of PBMC wasassessed by tritiated thymidine incorporation on days 6, 7, 8 and 9post-stimulation and stimulation indexes were calculated and plotted.

FIG. 3:

Frequency of observed responses with an SI>2 at any time point fromcohorts of 20 healthy donors to either wild type [Val¹ Val²]-hirudinregion 1 and modified R1 peptides.

FIG. 4:

Purification of [Val¹ Val²]-hirudin from transfected HEK.293 cellsupernatants. Elution of pooled fractions from pH 7.0 elution on Mono Qcolumn at pH 5.5. Main peak containing hirudin is marked with an arrow.

FIG. 5:

Silver stained SDS-PAGE gel of fractions from elution of Mono Q columnat pH 5.5. Lane 1=diluted supernatant loaded onto Mono Q column at pH7.0; Lane 2=Concentrated factions from elution of Mono Q column at pH7.0; Lane 3=Protein Standards; Lane 4=Fraction 1 pH 5.5 elution; Lane5=Fraction 10 pH 5.5 elution; Lane 6=Fraction 14 pH 5.5 elution; Lane7=Fraction 15 pH 5.5 elution; Lane 8=Fraction 17 pH 5.5 elution; Lane9=Fraction 19 pH 5.5 elution; Lane 10=Fraction 22 pH 5.5 elution; Lane11=Protein Standards. Bands corresponding to hirudin in lanes 6 and 7are marked with an arrow.

FIG. 6:

Thrombin inhibitory activity of the hirudin variants: I29A L30A and I29RL30H (M 1 and M 2 respectively). Cell-culture supernatants from I29AL30A(♦), I29R L30H (▪) and WT hirudin (▴) from transfected HEK.293cells, were compared in the chromogenic thrombin cleavage assay. Theamount of hirudin variants in tissue culture supernatants werequantified by ELISA, and the thrombin inhibition activity was titratedin 2 fold serial dilutions.

EXPERIMENTAL EXAMPLES Example 1 Construction of Hirudin Muteins

The cDNA for Hirudin was cloned from a cDNA library obtained by RT-PCRfrom mRNA extracted from the head of the medicinal leech, Hirudomedicinalis (Biopharm Ltd., Hendy, UK) using a Qiagen total RNAextraction kit (Qiagen, Crawley, UK) according to the manufacturer'sinstructions. This sequence was mutated to that of [Val¹ Val²]-hirudin,by site directed mutagenesis PCR [Higuchi et al (1988) Nucleic AcidsRes. 16: 7351].

For protein expression, the native 20 amino acid secretion signal,(MFSLKLFVVFLAVCICVSQA, SEQ ID NO: 85, of a hirudin variant from theAsian buffalo leech (Hirudinaria manillensis) was added by PCR and thisconstruct was then sub-cloned into the expression vector pREP4(Invitrogen, Paisley, UK). Later a modified secretion signal(MVSLKLFVVFLAVCICVSQA; SEQ ID NO: 86) was used in order to create a moreefficient Kozak consensus sequence for protein expression in mammaliancell lines [Kozak, M. (1987) Nucleic Acids Research; 15: 8125-8148;Kozak M. (1991) Journal of Cell Biology; 115: 887-903; Kozak, M. (1990)Proc. Natl. Acad. Sci. (USA); 87: 8301-8305], where the mutation of F²(TTC) to a V (GTC) placed a guanosine residue at position +4 in theconsensus sequence.

A total of 107 different hirudin muteins were constructed using PCRmutagenesis and the WT gene as a template. DNA sequencing was conductedon all constructs. This was diligently performed to confirm introductionof desired substitutions and establish that no extraneous (undesired)substitutions had been introduced for example by PCR error.

Example 2 Expression and Purification of Hirudin Proteins

Expression plasmids containing hirudin mutein genes were tranfected intothe human embryonic kidney cell line HEK.293. Transfection was conductedusing lipofectamine transfection reagent and cells were cultured in DMEMmedia free of phenol red (Invitrogen, Paisley, UK). Hirudin was purifiedfrom the culture media. Briefly, tissue culture supernatant was diluted1:1 with Mono Q buffer A (20 mM Bis-Tris-Propane pH 7.0). This was thenfiltered through a 0.22 μM membrane and applied to a Mono Q (HR5/5)column and protein was eluted using a 0-100% NaCl gradient in buffer A.Fractions were then tested for the presence of hirudin by ELISA.

Hirudin enriched fractions were pooled and concentrated using aCentricon centrifugal concentrator fitted with a YM3 membrane (3 kDa MWcut off). The pooled and concentrated fractions were buffer exchanged(G25 column) into buffer B (20 mM Piperazine pH 5.5) and, applied to aMono Q column equilibrated with buffer B. Protein was eluted with agradient from 0-100% 0.5M NaCl in buffer B and was monitored at 214 nm.Peak fractions were analysed on a 4-12% Bis-Tris SDS-PAGE gel(Invitrogen, Paisley, UK) using MES buffer. The gel was then silverstained to check the purity of the fractions.

Example 3 Detection and Quantitation of Hirudin Variants

Expressed hirudin was detected and quantitated using an ELISA with amatched pair of anti-hirudin antibodies [Koch C, et al (1993) AnalyticalBiochemistry; 214: 301-312]. Hirudin purified from Hirudo medicinalissaliva (Roche, Lewes, UK) was used as a standard in order to quantifyexpression. Briefly, a 96 well plate was coated with 100 μl of mouseanti-hirudin N-terminal epitope (Antibody shop, Copenhagen, Denmark) at10 μg/ml in PBS, overnight at 4° C. The plate was washed once withPBS/0.05% Tween, 200 μl per well, then blocked with 200 μl per well ofPBS/2% BSA for one hour at room temperature. The plate was then washed5× with PBS/0.05% Tween, 200 μl per well, then blotted onto pad of papertissues to remove excess liquid. A hirudin standard curve starting with1280 ng/ml was set up using doubling dilutions vertically down theplate. Hirudin variant samples were added in triplicate to a finalvolume of 100 μl in PBS at dilutions of 1:50 and 1:100. The plate wasincubated for 1 hr at room temperature then washed 5× as describedabove. 100 μl of biotinylated mouse anti-hirudin C-terminal epitope(Antibody Shop, Copenhagen, Denmark) was added at a 1:1000 dilution inPBS/2% BSA to each well and the plate incubated for 1 hr at roomtemperature. The plate was washed 5× as described above. 100 μl ofstreptavidin peroxidase (Sigma, Poole, UK) 1:1000 dilution in PBS/2% BSAwas added and the plate was incubated for 1 hr at room temperature. Theplate was washed as described above and 100 μl of o-phenylenediaminedihydrochloride substrate (Sigma, Poole, UK) was added to each well andthe plate incubated at room temperature for 5 minutes. The reaction wasquenched by adding 50 μl of 1M H₂SO₄ to each well and the absorbancemeasured at 492 nm.

Example 4 Functional Activity of Hirudin Muteins

The functional activity of the modified hirudin proteins was assessedusing a chromogenic thrombin inhibition assay. In this assay, a fixedamount of thrombin is incubated with a range of hirudin concentrationsat 37° C. The chromogenic substrate H-D-Phe-Pep-Arg-pNA is then addedand any residual free thrombin present will cleave the substrate to formH-D-Phe-Pep-Arg-OH and pNA that can then be detectedspectrophotometrically at 405 nm

Briefly, 2 ml of dH₂O was added to the thrombin vial and theH-D-Phe-Pep-Arg-pNA substrate vial from a Diagnostica Stago StachromHCII assay kit (Axis Shield Diagnostics, Dundee, UK) and they were bothwarmed in an incubator to 37° C. Hirudin assay buffer (175 mM NaCl, 7.5mM Na₂ EDTA, 50 mM Tris-HCl pH 8.4) was warmed to 37° C. then used todilute the test hirudin protein variants, hirudin wild type and hirudincontrol (Roche, Lewes, UK) proteins.

Aliquots of 40 μl of warm hirudin assay buffer were pipetted into wellsA-H 2-12 of a clear 96 well flat bottom plate, then 40 μl aliquots ofhirudin samples were placed into the wells in columns 1 and 2. Using amultichannel mix by pipetting up and down the contents of wells incolumn 2 and prepare a serial doubling dilution of the samples were madehorizontally across the plate by removing 40 μl from column 2 and addingto column 3 etc.

The plate was warmed to 37° C. for 5 minutes followed by addition of 40μl thrombin and incubated for 120 seconds at 37° C. with shaking. 40 μlH-D-Phe-Pep-Arg-pNA substrate was added, the plate and incubated for 90seconds at 37° C. with shaking. 40 μl acetic acid was added to quenchreaction and the plate was read spectrophotometrically at 405 nm.Absorbance at 405 nm vs hirudin concentration was plotted using Sigmaplot (Sigma Poole, UK) and IC₅₀ values calculated by fitting a sigmoidalequation.

A total of 81 different hirudin variants demonstrated positive activityin the chromogenic thrombin inhibition assay. Others either failed toexpress or showed no acceptable degree of activity. Positive activitywas taken to be a relative activity value of less than 10. Relativeactivity was determined by dividing the ED₅₀ value derived for theprotein of interest by the ED₅₀ value derived for the control (WT)protein.

Of these active proteins, 45 were muteins comprising a single amino acidsubstitution; 27 comprised two amino acid substitutions and 9 comprisedthree amino acid substitutions. The sequence of each of these activehirudin muteins is provided in M 1-81. The relative activities of eachfunctioning mutein are provided in Table 2 in which the heading labeled“Substitution” refers to substitutions in SEQ ID NO: 3.

A number of muteins show improved activity relative to the WT molecule.All active muteins are embodiments of the invention.

TABLE 2 Activity of hirudin variants Substitution Relative Activity*Substitution Relative Activity* L13T 0.60 D53E 1.0 L15A 0.90 D53N 1.0L15H 0.65 D53R 1.0 L15Q 0.34 F56H 0.80 L15T 0.40 I29A L3QA 1.40 V21A0.93 I29A L30K 1.45 V21G 1.0 I29A L30Q 1.60 V21H 0.74 I29A L30R 2.70V21K 0.90 I29A L30T 1.21 V21N 0.83 I29D L30A 1.60 V21P 1.0 I29D L30Q 2.0V21Q 0.83 I29D L30R 2.5 V21R 0.77 I29E L30K 1.10 I29A 1.0 I29E L30Q 1.52I29D 1.0 I29E L30R 1.21 I29E 1.0 I29E I30T 1.80 I29G 1.8 I29R L30H 1.2I29H 1.0 I29R L30K 0.93 I29K 1.0 I29R L30Q 1.0 I29N 1.0 I29R L30R 1.0I29Q 1.0 I29R L30T 1.0 I29R 1.0 I29S L30A 1.34 I29S 1.0 I29S L30K 1.50I29T 1.0 I29S L30Q 2.0 L30A 1.0 I29S L30R 2.5 L30D 2.0 I29S L30T 1.10L30E 3.33 I29T L30A 1.67 L30G 2.20 I29T L30K 1.60 L30H 1.0 I29T L30Q 2.4L30K 1.0 I29T L30R 2.30 L30N 1.54 I29T L30T 1.80 L30P 1.83 I29R L30K0.20 L30Q 1.0 I29R L30K 0.36 L30R 1.0 I29R L30R 0.70 L30S 1.83 I29E L30K1.0 L30T 1.0 I29E L30K 1.0 V40A 1.0 I29E L30K 0.55 V40T 1.0 I29E L30K1.0 K47T 2.2 I29R L30K 0.70 P48A 1.0 I29R L30K 0.80 P48T 1.0 *Ratio ofvariant IC₅₀/WT IC₅₀

Example 5 Identification of T-Cell Epitopes in Hiridin□

All blood samples used in this study were obtained with approval of theAddenbrooke's Hospital Local Research Ethics Committee. T-cell epitopemapping was performed using human PBMCs isolated from blood obtainedfrom the National Blood Transfusion Service (Addenbrooke's Hospital,Cambridge, UK). PBMCs from 25 healthy donors were isolated by Ficolldensity centrifugation and stored under liquid nitrogen. Each donor wastissue-typed using an Allset™ PCR based tissue-typing kit (Dynal) and Tcell assays were performed by selecting donors according to individualMHC haplotypes. 15 mer peptides staggered by three amino acids andspanning the human hirudin sequence were purchased from Pepscan SystemsBV (NL). Using this scheme, total of 19 peptides were required to scanthe hirudin protein. The sequence and peptide number of these peptidesare provided in Table 3.

TABLE 3 Peptides used to map immunogenic epitopes within hirudin PeptideSEQ ID No NO: Peptide sequence 1 87 VVYTDCTESGQNLCL 2 88 LTYTDCTESGQNLCL3 89 TDCTESGQNLCLCEG 4 90 TESGQNLCLCEGSNV 5 91 GQNLCLCEGSNVCGQ 6 92LCLCEGSNVCGQGNK 7 93 CEGSNVCGQGNKCIL 8 94 SNVCGQGNKCILGSD 9 95CGQGNKCILGSDGEK 10 96 GNKCILGSDGEKNQC 11 97 CILGSDGEKNQCVTG 12 98GSDGEKNQCVTGEGT 13 99 GEKNQCVTGEGTPKP 14 100 NQCVTGEGTPKPESH 15 101VTGEGTPKPESHNDG 16 102 EGTPKPESHNDGDFE 17 103 PKPESHNDGDFEEIP 18 104ESHNDGDFEEIPEEY 19 105 NDGDFEEIPEEYYLQ

For each donor sample, PBMCs were thawed and resuspended in AIM-V(Invitrogen) containing 100 units/ml penicillin, 100 μg/ml streptomycinand 1 mM glutamine. Triplicate cultures of 2×10⁵ PBMC/well offlat-bottomed 96 well plate were incubated with peptides at a finalconcentration of 1 μM and 10 μM. Cells were incubated for 7 days beforepulsing with 1 μCi/well tritiated thymidine for 18 hours. Cultures wereharvested onto glass fibre filter mats using a Tomtec Mach III plateharvester and cpm values determined by scintillation counting usingWallac Microbeta TriLux plate reader.

Each donor was also tested for their ability to respond to two positivecontrol peptides influenza haemagglutinin A amino acids 307-319 [KriegerJ I, et al (1991) Journal of Immunology; 146: 2331-2340] and chlamydiaHSP60 amino acids 125-140 [Cerrone M C, et al (1991) Infection andImmunity; 59: 79-90]. Keyhole limpet haemocyanin, a well documentedpotent T cell antigen was also used as a control.

Donors that responded to peptides with an SI>2 were analyzed further byplotting the frequency of donor responses to each peptide. Prominentregions of immunogencity were determined by peptides that inducedresponses in 10% of donors; however, borderline responses whereindividual SI values >1.95 were achieved and if two adjacent peptidesinduced responses in 5% of donors.

In the initial analysis using 25 donor samples, peptides located withintwo separate regions were able to induce T cell proliferation R1 waslocated at peptide 10 where donor response was 8% and R2 located atpeptide 15 where the donor response was also 8%. Subsequent analysisusing further 30 donor samples failed to maintain the 8% donor responserate for peptide 15 within R2 epitope region (see example 6). The finalresponse rate across a total of 55 donor samples for peptide 15 was 4%(2/55) and R2 was not considered a significant epitope. FIG. 1 shows themature sequence of [Val¹ Val²]-hirudin with regions of immunogenicityhighlighted in bold.

Example 6 Analysis of Immunogenic Regions by Time-Course T-Cell Assays

Bulk cultures of 2-4×10⁶ PBMC/well were established from 20 healthydonor samples in 24 well plates. Cells were incubated for 6 to 9 dayswith WT and variant peptides spanning the immunogenic regions (see Table4). T cell proliferation was assessed by tritiated thymidineincorporation on days 6, 7, 8 and 9. Proliferation was assessed at eachtime point, by gently resuspending the bulk cultures and removingsamples of PBMC, that were then incubated in triplicate wells ofU-bottomed 96 well plate with 1 μCi/well tritiated thymidine for 18hours as described above.

The time course assay was used to test variant peptides containingsubstitutions over WT. Substitutions were made at key locations wherethere was expectation that the substitution would prevent binding to MHCclass II and therefore, subsequent T cell proliferation in the assay.FIG. 2 and FIG. 3 respectively show exemplary donor responses over thetime course and overall response frequencies. Table 4 shows thesequences of the peptides tested and also the overall responsefrequencies to each peptide.

The frequency of response is significantly reduced for all substitutionstested. The most effective substitutions are I29A L30A (M 1) and I29RL30H (M 2).

TABLE 4 Sequences of peptides used in time-course assays % donor % donorresponse response Wild Type in time Modified in time Sequence courseSequences course CILGSDGEKNQCVTG 25 CAAGSDGEKNQCVTG 5 SEQ ID NO: 97 SEQID NO: 106 CEKGSDGEKNQCVTG 10 SEQ ID NO: 107 CRHGSDGEKNQCVTG 5 SEQ IDNO: 108

Example 7 Functional Activity of Most Preferred Hirudin Muteins

The thrombin inhibitory activity of the most preferred molecules of theinvention was tested. Results are shown in Table 5. Cell-culturesupernatants from variants and WT hirudin from transfected HEK.293cells, were compared in the chromogenic thrombin cleavage assay. Theamount of hirudin variants in tissue culture supernatants werequantified by ELISA, and the thrombin inhibition activity was titratedin 2 fold serial dilutions.

The most preferred molecules of the invention demonstrate functionalactivity within 60% of the WT molecule.

TABLE 5 Functional activity of most preferred hirudin muteins Relativeactivity Hirudin variant (ED₅₀ variant/ED₅₀ WT hirudin) I29A L30A (M 1)1.4 I29R L30H (M 2) 1.2

1. An isolated peptide consisting of the amino acid residue sequenceCILGSDGEKNQCVTGEGTPKPESHNDGDFE (SEQ ID NO: 1).
 2. A pharmaceuticalcomposition comprising the peptide of claim 1, optionally together witha pharmaceutically acceptable carrier, diluent or excipient.